Two single strands of deoxyribo-("DNA") or ribo("RNA") nucleic acid, comprised of nucleotides (adenine, cytosine, thymidine, guanine, uracil, inosine, etc.), may associate ("hybridize") to form a double helical structure in which the two polynucleotide chains running in opposite directions are held together by hydrogen bonds (a weak form of chemical bond) between pairs of matched, centrally located compounds known as "bases." Generally, in the double helical structure of nucleic acids, the base adenine (A) is hydrogen bonded to the base thymine (T) or uracil (U) while the base guanine (G) is hydrogen bonded to the base cytosine (C). At any point along the chain, therefore, one may find the classical "Watson-Crick" base pairs AT or AU, TA or UA, GC, or CG. One may also find AG, GU and other "wobble" or mismatched base pairs in addition to the traditional ("canonical") base pairs. Assuming that a first single strand of nucleic acid is sufficiently complementary to a second and that the two are brought together under conditions which will promote their hybridization, double stranded nucleic acid will result. Under appropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids may be formed.
A probe may be a single strand nucleic acid sequence which is complementary in some particular degree to the nucleic acid sequences sought to be detected ("target sequences"). It may also be labelled with a detectable moiety such as a radio-isotope, antigen or chemiluminescent moiety. A background description of the use of nucleic acid hybridization as a procedure for the detection of particular nucleic acid sequences is described in U.S. Pat. No. 4,851,330 to Kohne and entitled "Method for Detection, Identification and Quantitation of Non-Viral Organisms," issued Jul. 25, 1989 and in EPO Application No. PCT/US87/03009 to Hogan et al., entitled "Nucleic Acid Probes for Detection and/Or Quantitation of Non-Viral Organisms."
Also described in the Kohne patent and the Hogan et al. application are methods for determining the presence of RNA-containing organisms in a sample which might contain such organisms. These methods require the mixture of nucleic acids from a sample and a probe comprised of nucleic acid molecules which are shorter than the ribosomal-RNA ("rRNA") subunit sequence from which it was derived. The probes are sufficiently complementary to hybridize to the rRNA of one or more non-viral organisms or groups of non-viral organisms. The mixture is then incubated under specified hybridization conditions, and assayed for hybridization of the probe and any test sample rRNA.
Further, the Hogan et al. application describes numerous probes which detect only specifically targeted rRNA subunit subsequences in particular organisms or groups of organisms in a sample, even in the presence of many non-related organisms, or in the presence of closest known phylogenetic neighbors. The Hogan et al. application discloses hybridization assay probes for Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti, the genus Mycobacterium, Mycoplasma pneumoniae, the genus Legionella, Chlamydia trachomatis, the genus Campylobacter, Enteroccoccus, the genus Pseudomonas group I, Enterobacter cloacae, Proteus mirabilis, the genus Salmonella, Escherichia coli, bacteria, fungi, and Neisseria gonorrhoeae. Such probe sequences do not cross react with nucleic acids from the groups listed above, or any other bacterial species or infectious agent, under proper stringency.
This invention discloses and claims novel probes for the detection of Haemophilus influenzae. These probes are capable of distinguishing between Haemophilus influenzae and its known closest phylogenetic neighbors.